Homodimeric protein constructs

ABSTRACT

The present invention relates to novel recombinant fusion proteins, such as human antibody-based molecules called Vaccibodies, which are able to trigger both a T cell- and B cell immune response. The present invention also relates to a method of treating a cancer or an infectious disease by means of these specific fusion proteins.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/784,778, filed Feb. 7, 2020, which is a divisional of U.S.application Ser. No. 13/805,709, filed Jan. 25, 2013, which is anational stage filing under 35 U.S.C. 371 of PCT/EP2011/060628, filedJun. 24, 2011, which International Application was published by theInternational Bureau in English on Jun. 24, 2011, and application claimspriority from U.S. Application No. 62/358,513, filed Jun. 25, 2010, andEuropean Application No. 10167291.3, filed Jun. 25, 2010 whichapplications are hereby incorporated in their entirety by reference inthis application.

SEQUENCE LISTING

This application contains a Sequence Listing which is submitted herewithin electronically readable format. The Sequence Listing file was createdon Sep. 21, 2022, is named V89540_0014_2_Seq_List.xml, and is 14.4 KB insize. The entire contents of the Sequence Listing in the sequencelisting .xml file are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to novel recombinant fusion proteins, suchas human antibody-based molecules called Vaccibodies, which are able totrigger both a T cell- and B cell immune response. The present inventionalso relates to a method of treating a cancer or an infectious diseasee.g. multiple myeloma or influenza by means of these specific fusionproteins.

BACKGROUND OF THE INVENTION

DNA vaccination is a technically simple way of inducing immuneresponses. However, success in small animals has not yet been reproducedin clinical trials. Several strategies are currently being pursued toincrease efficacy of DNA vaccines.

Targeting of protein antigens to antigen-presenting cells (APC) canimprove T- and B-cell responses. Recombinant immunoglobulin (Ig)molecules are well suited for this purpose. For example, short antigenicepitopes can replace loops between β-strands in the Ig constant domainswhile targeted antigen delivery is obtained by equipping the recombinantIg with variable (V) regions specific for surface molecules on APC.However, such a strategy is unfit for larger antigens containingunidentified epitopes, moreover recombinant Ig molecules with short Tcell epitopes fail to elicit antibodies against conformational epitopes.To overcome these limitations, targeted Ig-based homodimeric DNAvaccines (vaccibodies) have been generated that express infectious ortumor antigens with a size of at least 550 aa. with maintenance ofconformational epitopes.

Chemokine (C—C motif) ligand 3 (CCL3) is a protein that in humans isencoded by the CCL3 gene. CCL3, also known as Macrophage inflammatoryprotein-1α (MIP-1α), is a cytokine belonging to the CC chemokine familythat is involved in the acute inflammatory state in the recruitment andactivation of polymorphonuclear leukocytes. While mouse CCL3 is a singlecopy gene encoding for a mature chemokine of 69 amino acids, the humanhomolog has been duplicated and mutated to generate two non-allelicvariants, LD78α (CCL3) and LD78β (CCL3-L1), both showing a 74% homologywith the mouse CCL3.

No DNA vaccine has so far been approved for human use due to lack ofefficacy. Also there is no effective vaccine available for severalinfectious diseases. In particular, no therapeutic DNA cancer vaccinehas been approved for human use.

WO 2004/076489 relates to recombinant human antibody-based moleculecalled Vaccibodies, which are able to trigger both a T cell- and B cellimmune response.

US20070298051 relates to the use of MIP-1-alpha for enhancing the immuneresponse to an immunogen in a mammal.

EP920522 relates to a polynucleotide vector vaccine comprising a cDNAtarget product that comprises a nucleotide sequence encoding a cytokineor chemokine.

Fredriksen A B et al. (Mol Ther 2006; 13:776-85) relates to DNA vaccinestargeting tumor antigen to antigen-presenting cells.

Fredriksen A B and Bogen B (Blood 2007; 110: 1797-805) relates to mousechemokine-idiotype fusion DNA vaccines.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide fusionproteins, which are able to trigger an efficient immune response foreven weak antigens, such as idiotypic antigens derived from e.g. myelomacells.

Furthermore it is an object of embodiments of the invention to providepolynucleotides, such as a DNA polynucleotide, encoding a fusion proteinthat trigger an efficient immune response against even weak antigens,such as idiotypic antigens derived from e.g. myeloma cells. Thesepolynucleotides may be used as an immunostimulating composition orvaccine against a cancer or an infectious disease, characterized by adisease specific or disease associated antigen.

SUMMARY OF THE INVENTION

It has been found by the present inventor(s) that human chemokine LD78β,both full length and truncated versions thereof, are suited for use astargeting units that target antigenic epitopes to the surface of APC.The chemokine, or its truncated version are bound to chemokine receptorson the surface of APC in the form of a homodimeric protein construct,which facilitate that two identical chemokines are bound to provide moreefficient targeting and signalling. The homodimeric construct furtherprovide that two identical antigenic epitopes are delivered to the APCwhich in turn present them to T cells. Even with the relatively largesize of the homodimeric protein constructs, cells are able to produceand export intact molecules.

So, in a first aspect, the present invention relates to a homodimericprotein of two identical amino acid chains, each amino acid chaincomprising a targeting unit comprising an amino acid sequence having atleast 80% sequence identity to the amino acid sequence 5-70 of SEQ IDNO: 1, and an antigenic unit, the targeting unit and the antigenic unitbeing connected through a dimerization motif.

In a second aspect, the present invention relates to a homodimericprotein of two identical amino acid chains, each amino acid chaincomprising a targeting unit comprising amino acids 3-70 of SEQ ID NO: 1,and an antigenic unit, the targeting unit and the antigenic unit beingconnected through a dimerization motif.

In a third aspect, the present invention relates to a nucleic acidmolecule encoding the monomeric protein which can form a homodimericprotein according to the invention.

In a further aspect, the present invention relates to a homodimericprotein according to the invention; for use as a medicament.

In a further aspect, the present invention relates to a nucleic acidmolecule encoding the monomeric protein which can form a homodimericprotein according to the invention; for use as a medicament.

In a further aspect, the present invention relates to a pharmaceuticalcomposition comprising a homodimeric protein according to the invention.

In a further aspect, the present invention relates to a pharmaceuticalcomposition comprising a nucleic acid molecule encoding the monomericprotein which can form a homodimeric protein according to the invention.

In a further aspect, the present invention relates to a host cellcomprising a nucleic acid molecule encoding the monomeric protein whichcan form a homodimeric protein according to the invention.

In a further aspect, the present invention relates to a method forpreparing a homodimeric protein according to the invention, the methodcomprising

-   -   a) transfecting the nucleic acid molecule according to the        invention into a cell population;    -   b) culturing the cell population;    -   c) collecting and purifying the homodimeric protein expressed        from the cell population.

In a further aspect the present invention relates to a vaccine against acancer or an infectious disease comprising an immunologically effectiveamount of a homodimeric protein according to the invention or nucleicacid molecule encoding the monomeric protein which can form thehomodimeric protein according to the invention, wherein said vaccine isable to trigger both a T-cell- and B-cell immune response and whereinsaid homodimeric protein contain an antigenic unit related to saidcancer or infectious disease.

In a further aspect the present invention relates to an immunomodulatingor immunostimulating composition against a cancer or an infectiousdisease comprising an immunologically effective amount of a homodimericprotein according to the invention or nucleic acid molecule encoding themonomeric protein which can form the homodimeric protein according tothe invention, wherein said immunomodulating or immunostimulatingcomposition is able to trigger both a T-cell- and B-cell immune responseand wherein said homodimeric protein contain an antigenic unit relatedto said cancer or infectious disease.

In a further aspect the present invention relates to a method oftreating a cancer or an infectious disease in a patient, the methodcomprising administering to the patient in need thereof, a homodimericprotein according to the invention, or the nucleic acid moleculeencoding the monomeric protein which can form the homodimeric proteinaccording to the invention, wherein said homodimeric protein contain anantigenic unit related to said cancer or infectious disease.

LEGENDS TO THE FIGURE

FIG. 1 . Fusion vaccines used in this study. (A) Schematic structure ofa homodimeric chemokine-antigen fusion protein (vaccibody). Targeting,dimerization and antigenic units are indicated as are moieties expressedin the various units. In all constructs, the dimerization unit and hingeare derived from human IgG3. A G₃S₂G₃SG linker connects hinge exonsh1+h4 to the CH3 domain. A GLSGL linker connects CH3 and the antigenicunit, whereas a (G₄S)₃ linker connects V_(H) and V_(L) in the antigenicunit. (B) NH2 terminal sequences (aa. 1-12) of human CCL3 isoforms, andtheir point mutated control (C11S, indicated in bold). Slash indicatesdeletion. (C) The C11S point mutation putatively destroys a S—S bridgein the chemokine structure (right).

FIG. 2 . Characterization of LD78β-expressing vaccibodies by ELISA andWestern blot. Supernatants of transiently transfected 293E cellscollected at day 5 were tested in ELISA by using mAbs specific fordifferent components of the vaccibody molecules. (A), scFv³¹⁵ encodingvaccibodies with indicated targeting units were evaluated by binding toDNP-BSA coat (binds scFv³¹⁵) and detection with biotinylated HP6017(binds CH3 dimerization motif). (B), CκCκ-encoding vaccibodies, withindicated targeting units, were evaluated by binding to 187.1 mAb (bindsmouse Cκ) coat and detection with biotinylated 187.1 mAb, (C),HA-encoding vaccibodies, with indicated targeting units, were evaluatedby binding to MCA878-G (anti-CH3 dimerization motif) and detection withbiotinylated anti-HA mAb H36-4-52 (D), Western blot of vaccibodiesprobed with biotinylated HP6017 under non-reducing conditions. Left toright, (LD78βFv³¹⁵)2, (LD78βC11SFv³¹⁵)2 and (LD78β-2Fv³¹⁵)2.

FIG. 3 . LD78β vaccibody proteins bind chemokine receptors on humancells. The indicated homodimeric vaccibody proteins at 25 μg/mL wereadmixed with HEK 293 cells stably transfected with either human CCR5 (A,B), or human CCR1 (C, D). Bound vaccibody proteins were detected bybiotinylated Ab2.1-4 mAb specific for the scFv³¹⁵ antigenic unit,followed by PE-streptavidin. Bold lines: (LD78βFv³¹⁵)2 (A, C) and(LD78β-2Fv³¹⁵)2 (B, D) vaccibodies. Dashed line in (A):(LD78β(C11S)Fv³¹⁵)2. Shaded histogram: biotinylated Ab2.1-4 mAb andPE-streptavidin alone.

FIG. 4 . LD78β vaccibody proteins bind mouse chemokine receptors andinduce chemotaxis of murine cells. (LD78βFv³¹⁵)2 vaccibody (openhistogram), but not the C11S variant (shaded histogram), binds to CD11b+BALB/c splenocytes (A) and displays chemotactic activity on lymphocyticEsb/MP cells (B).

FIG. 5 . Vaccibody with LD78β targeting unit efficiently deliversantigen to mouse (A) and human (B) APC for MHC class II-restrictedpresentation to CD4+ T cells. (A) Different amounts of purifiedvaccibodies having scFv³¹⁵ as antigenic unit were admixed withirradiated (8 gy) BALB/c splenocytes, followed by addition ofId(λ2³¹⁵)-specific Th2 T cells from TCR transgenic mice. After 48 hrscultures were pulsed with 3H thymidine for 24 hrs. (B) Different amountsof mouse Cκ-containing vaccibody supernatants (expressed as molarconcentration (M) of CκCκ) from transiently transfected 293E cells wereadmixed with DR4*01 PBMCs which were then irradiated and admixed withmouse Cκ-specific T18 T cells. After 48 hrs the plate was pulsed with 3Hthymidine for 24 hrs.

FIG. 6 . Anti-Id³¹⁵ immune responses in mice immunized with LD78βFv³¹⁵vaccibody DNA. Mice were immunized by intradermal administration of DNAimmediately followed by electroporation of the injection site. Type ofvaccibodies and controls are indicated. Sera obtained 3 weeks later weretested for anti-Id IgG1 (A) or IgG2a (B) antibodies binding the M315myeloma protein. Mean of up to 7 mice per group is shown. p values referto LD78β vs LD78β (C11S) (*), and to LD78β vs (FvNIP)2 vaccibody (**) atweek 4.

FIG. 7 . Induction of CD4+ and CD8+ influenza hemagglutinin-specific Tcell responses by LD78β-vaccibodies. Mice (n=3) were immunized byintradermal administration of DNA immediately followed byelectroporation of the injection site (Dermavax, Cytopulse, USA). Typeof vaccibodies and controls are indicated. Mice were sacrificed 3 weekslater and individual splenocyte suspensions used in ELISPOT assays withthe indicated MHC class II- and class I-restricted synthetic HApeptides, or irrelevant peptide. IFNγ responses were evaluated. p valuesrefer to LD78β vs LD78βC11S and LD78β vs 0.9% NaCl (*), and to LD78βC11Svs 0.9% NaCl (**).

FIG. 8 . LD78β vaccibodies binds to rhesus macaque CCR5. Vaccibodyproteins at 25 μg/mL were admixed with HEK 293 stably transfected withRhesus macaque CCR5. Bound vaccibody proteins were detected bybiotinylated Ab2.1-4 mAb specific for the scFv³¹⁵ antigenic unitfollowed by PE-streptavidin. Bold line indicates vaccibodies(LD78βFv³¹⁵)2 in (A) and (LD78β-2Fv³¹⁵)2 in (B). Dashed line in (A)indicates (LD78β(C11S)Fv³¹⁵)2 vaccibody. In both A and B shadedhistograms indicate biotinylated Ab2.1-4 mAb and PE-streptavidin alone.

FIG. 9 . Protection against a lethal challenge with influenza. Balb/cmice were immunized once intradermally with 25 μg DNA in combinationwith electroporation (DermaVax), and challenged after 14 days(n=6/group) with a lethal dose of PR8 influenza virus (H1N1).

DETAILED DISCLOSURE OF THE INVENTION

Efficacy of DNA vaccines needs to be increased. A promising strategy inmice is to construct DNA encoding a fusion protein that target antigento antigen-presenting cells (APC) via chemokine receptors. It is crucialto extend this strategy for improved DNA vaccines to large animals andhumans. According to the present invention, human MIP-1α chemokines maybe fused with different antigenic units. The fusion proteins retainfunctional activity and conformational correctness of targeting andantigenic units, respectively. Fusion proteins may improve responses ofcloned human CD4+ T cells. Moreover, since LD78β fusion proteins bindsmouse chemokine receptors, human DNA vaccines can be tested in mice.LD78β DNA fusion vaccines according to the present invention inducedimproved T cell and antibody responses in mice following plasmidinjection and skin electroporation. CD8+ T cell responses areparticularly enhanced, indicating efficient cross-priming. A two aminoacid NH₂-truncated version of LD78β is by the present inventors provedto have superior binding to mouse cells compared to the full lengthLD78β in vitro. Surprisingly the full length version of LD78β haveshowed superior effect in an in vivo mouse model. LD78β-vaccine proteinswas found by the inventors of the present invention to bind Rhesusmacaque CCR5, setting the stage for targeted DNA immunization in nonhuman primates.

Vaccibodies according to the present invention may be recombinantIg-based homodimeric vaccines, each chain being composed of a targetingunit directly attached to Ig hinge and CH3, the combination of whichinduces covalent homodimerization (FIG. 1A).

While mouse CCL3 is a single copy gene encoding for a mature chemokineof 69 aa., the human homolog has been duplicated and mutated to generatetwo non-allelic variants, LD78α (CCL3) and LD78β (CCL3-L1), both showinga 74% homology with the mouse CCL3. The two variants share a 96%homology, the differences being S or P at position 2 and a swap betweenG and S at positions 39 and 47.

According to the present invention, human CCL3 variants and differentantigenic units, may be constructed and expressed as functionalproteins. In particular, the present invention relates to theutilization of LD78β and its natural isoforms in fusion vaccines totarget antigen delivery to antigen-presenting cells.

The vaccibodies according to the present invention aims at improving theimmunogenicity of vaccines (immunostimulating compositions). Includedwithin the present invention are DNA vaccines encoding a fusion proteinthat targets antigen delivery to LD78β receptors on professionalantigen-presenting cells.

Vaccibodies equipped with LD78β or NH₂-truncated versions hereof were bythe inventors of the present invention found to bind cells expressingmurine or Rhesus macaque or human CCR1 and/or CCR5 (receptors for LD78β)in vitro and afforded augmented antigen delivery in vitro as well asincreased humoral and cellular immune responses in vivo following DNAinjection and electroporation, as compared with control, non-targetedvaccibodies.

The recombinant proteins according to the present invention may be humanantibody-like molecules useful in the treatment of many types of canceror infectious diseases, including multiple myeloma. These molecules,also referred to as Vaccibodies, bind APC and are able to trigger both Tcell and B cell immune response. Moreover, Vaccibodies bind divalentlyto APC to promote a more efficient induction of a strong immuneresponse. Vaccibodies comprise a dimer of a monomeric unit that consistsof a targeting unit with specificity for a surface molecule on APC,connected through a dimerization motif, such as a hinge region and a Cy3domain, to an antigenic unit, the later being in the COOH-terminal orNH2-terminal end. The present invention also relates to a DNA sequencecoding for this recombinant protein, to expression vectors comprisingthese DNA sequences, cell lines comprising said expression vectors, totreatment of mammals preferentially by immunization by means ofVaccibody DNA, Vaccibody RNA, or Vaccibody protein, and finally topharmaceuticals and a kit comprising the said molecules.

The dimerization motif in the proteins according to the presentinvention may be constructed to include a hinge region and animmunoglobulin domain (e.g. Cy3 domain), e.g. carboxyterminal C domain(C_(H)3 domain), or a sequence that is substantially homologous to saidC domain. The hinge region may be Ig derived and contributes to thedimerization through the formation of an interchain covalent bond(s),e.g. disulfide bridge(s). In addition, it functions as a flexible spacerbetween the domains allowing the two targeting units to bindsimultaneously to two target molecules on APC expressed with variabledistances. The immunoglobulin domains contribute to homodimerizationthrough noncovalent interactions, e.g. hydrophobic interactions. In apreferred embodiment the C_(H)3 domain is derived from IgG. Thesedimerization motifs may be exchanged with other multimerization moieties(e.g. from other Ig isotypes/subclasses). Preferably the dimerizationmotif is derived from native human proteins, such as human IgG.

It is to be understood that the dimerization motif may have anyorientation with respect to antigenic unit and targeting unit. In oneembodiment the antigenic unit is in the COOH-terminal end of thedimerization motif with the targeting unit in the N-terminal end of thedimerization motif. In another embodiment the antigenic unit is in theN-terminal end of the dimerization motif with the targeting unit in theCOOH-terminal end of the dimerization motif. International applicationWO 2004/076489, which is hereby incorporated by reference disclosesnucleic acid sequences and vectors, which may be used according to thepresent invention.

The proteins according to the present invention may be suitable forinduction of an immune response against any polypeptide of any origin.Any antigenic sequence of sufficient length that include a specificepitope may be used as the antigenic unit in the proteins according tothe invention. The minimal length of such antigenic unit may be around 9amino acids. Accordingly in some embodiments, the antigenic unitcomprises an amino acid sequence of at least 9 amino acids correspondingto at least about 27 nucleotides in a nucleic acids sequence encodingsuch antigenic unit. Such an antigenic sequence may be derived fromcancer proteins or infectious agents. Examples of such cancer sequencesare telomerase, more specifically hTERT, tyrosinase, TRP-1/TRP-2melanoma antigen, prostate specific antigen and idiotypes. Theinfectious agents can be of bacterial, e.g. tuberculosis antigens andOMP31 from brucellosis, or viral origin, more specifically HIV derivedsequences like e.g. gp120 derived sequences, glycoprotein D from HSV-2,and influenza virus antigens like hemagglutinin, nucleoprotein and M2.Insertion of such sequences in a Vaccibody format might also lead toactivation of both arms of the immune response. Alternatively theantigenic unit may be antibodies or fragments thereof, such as theC-terminal scFv derived from the monoclonal Ig produced by myeloma orlymphoma cells, also called the myeloma/lymphoma M component in patientswith B cell lymphoma or multiple myeloma. Such scFv represents idiotypicantigen.

In one particular embodiment, also used in the examples describedherein, the antigenic unit of the protein according to the presentinvention is the scFv of the myeloma protein M315 derived from theBALB/c plasmacytoma MOPC315.4. The λ2³¹⁵ light chain of M315 harborsthree defined somatic mutations in the CDR3 loop and functions as amodel idiotypic T cell epitope in a well defined system (Bogen, Malissenet al. 1986; Bogen and Lambris 1989).

Immunization by means of Vaccibody protein, Vaccibody DNA, or VaccibodyRNA, the latter two executed e.g. by intramuscular or intradermalinjection with or without a following electroporation, are all feasiblemethods.

The targeting unit of the proteins according to the invention targetsthe protein to APC through binding to chemokine receptors.

The proteins according to the present invention may be geneticallyassembled, and the DNA transfected into a suitable host cell, such asNS0 cells, 293E cells, CHO cells or COS-7 cells. Transfectants produceand secrete the recombinant proteins.

The present invention relates to a pharmaceutical comprising the abovedescribed recombinant based proteins, DNA/RNA sequences, or expressionvectors according to the invention. Where appropriate, thispharmaceutical additionally comprises a pharmaceutically compatiblecarrier. Suitable carriers and the formulation of such pharmaceuticalsare known to a person skilled in the art. Suitable carriers are e.g.phosphate-buffered common salt solutions, water, emulsions, e.g.oil/water emulsions, wetting agents, sterile solutions etc. Thepharmaceuticals may be administered orally or parenterally. The methodsof parenteral administration comprise the topical, intra-arterial,intramuscular, subcutaneous, intramedullary, intrathekal,intraventricular, intravenous, intraperitoneal or intranasaladministration. The suitable dose is determined by the attendingphysician and depends on different factors, e.g. the patient's age, sexand weight, the kind of administration etc. Furthermore, the presentinvention relates to a vaccine composition or immunostimulatingcompositions against cancer or infectious diseases comprising animmunologically effective amount of the nucleic acid encoding themolecule of the invention or degenerate variants thereof, wherein saidcomposition is able to trigger both a T-cell- and B-cell immuneresponse. The present invention also relates to a kit comprisingVaccibody DNA, RNA, or protein for diagnostic, medical or scientificpurposes.

The invention further relates to a method of preparing the recombinantmolecule of the invention comprising, transfecting the vector comprisingthe molecule of the invention into a cell population; culturing the cellpopulation; collecting recombinant protein expressed from the cellpopulation; and purifying the expressed protein.

The above described nucleotide sequences may preferably be inserted intoa vector suited for gene therapy, e.g. under the control of a specificpromoter, and introduced into the cells. In a preferred embodiment thevector comprising said DNA sequence is a virus, e.g an adenovirus,vaccinia virus or an adeno-associated virus. Retroviruses areparticularly preferred. Examples of suitable retroviruses are e.g.MoMuLV or HaMuSV. For the purpose of gene therapy, the DNA/RNA sequencesaccording to the invention can also be transported to the target cellsin the form of colloidal dispersions. They comprise e.g. liposomes orlipoplexes.

The present invention also encompasses the use of polypeptides ordomains or motifs within the polypeptides having a degree of sequenceidentity or sequence homology with amino acid sequence(s) defined hereinor with a polypeptide having the specific properties defined herein. Thepresent invention encompasses, in particular, peptides having a degreeof sequence identity with SEQ ID NO: 1, or homologues thereof. Here, theterm “homologue” means an entity having sequence identity with thesubject amino acid sequences or the subject nucleotide sequences, wherethe subject amino acid sequence preferably is SEQ ID NO: 1.

In one aspect, the homologous amino acid sequence and/or nucleotidesequence should provide and/or encode a polypeptide which retains thefunctional activity and/or enhances the activity of a polypeptide of SEQID NO: 1.

In the present context, a homologous sequence is taken to include anamino acid sequence which may be at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, identical to the subject sequence. Typically, the homologues willcomprise the same active sites etc. as the subject amino acid sequence.Although homology can also be considered in terms of similarity (i.e.amino acid residues having similar chemical properties/functions), inthe context of the present invention it is preferred to express homologyin terms of sequence identity.

Sequence identity comparisons can be conducted by eye, or more usually,with the aid of readily available sequence comparison programs. Thesecommercially available computer programs use complex comparisonalgorithms to align two or more sequences that best reflect theevolutionary events that might have led to the difference(s) between thetwo or more sequences. Therefore, these algorithms operate with ascoring system rewarding alignment of identical or similar amino acidsand penalising the insertion of gaps, gap extensions and alignment ofnon-similar amino acids. The scoring system of the comparison algorithmsinclude:

-   -   i) assignment of a penalty score each time a gap is inserted        (gap penalty score),    -   ii) assignment of a penalty score each time an existing gap is        extended with an extra position (extension penalty score),    -   iii) assignment of high scores upon alignment of identical amino        acids, and    -   iv) assignment of variable scores upon alignment of        non-identical amino acids.

Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons.

The scores given for alignment of non-identical amino acids are assignedaccording to a scoring matrix also called a substitution matrix. Thescores provided in such substitution matrices are reflecting the factthat the likelihood of one amino acid being substituted with anotherduring evolution varies and depends on the physical/chemical nature ofthe amino acid to be substituted. For example, the likelihood of a polaramino acid being substituted with another polar amino acid is highercompared to being substituted with a hydrophobic amino acid. Therefore,the scoring matrix will assign the highest score for identical aminoacids, lower score for non-identical but similar amino acids and evenlower score for non-identical non-similar amino acids. The mostfrequently used scoring matrices are the PAM matrices (Dayhoff et al.(1978), Jones et al. (1992)), the BLOSUM matrices (Henikoff and Henikoff(1992)) and the Gonnet matrix (Gonnet et al. (1992)).

Suitable computer programs for carrying out such an alignment include,but are not limited to, Vector NTI (Invitrogen Corp.) and the ClustalV,ClustalW and ClustalW2 programs (Higgins D G & Sharp P M (1988), Higginset al. (1992), Thompson et al. (1994), Larkin et al. (2007). A selectionof different alignment tools is available from the ExPASy Proteomicsserver at www.expasy.org. Another example of software that can performsequence alignment is BLAST (Basic Local Alignment Search Tool), whichis available from the webpage of National Center for BiotechnologyInformation which can currently be found at http://www.ncbi.nlm.nih.gov/and which was firstly described in Altschul et al. (1990) J. Mol. Biol.215; 403-410.

Once the software has produced an alignment, it is possible to calculate% similarity and % sequence identity. The software typically does thisas part of the sequence comparison and generates a numerical result.

In one embodiment, it is preferred to use the ClustalW software forperforming sequence alignments. Preferably, alignment with ClustalW isperformed with the following parameters for pairwise alignment:

Substitution matrix: Gonnet 250 Gap open penalty: 20 Gap extensionpenalty: 0.2 Gap end penalty: None

ClustalW2 is for example made available on the internet by the EuropeanBioinformatics Institute at the EMBL-EBI webpage www.ebi.ac.uk undertools—sequence analysis—ClustalW2. Currently, the exact address of theClustalW2 tool is www.ebi.ac.uk/Tools/clustalw2.

In another embodiment, it is preferred to use the program Align X inVector NTI (Invitrogen) for performing sequence alignments. In oneembodiment, Exp10 has been may be used with default settings:

Gap opening penalty: 10Gap extension penalty: 0.05Gap separation penalty range: 8Score matrix: blosum62mt2

Thus, the present invention also encompasses the use of variants,homologues and derivatives of any amino acid sequence of a protein,polypeptide, motif or domain as defined herein, particularly those ofSEQ ID NO: 1.

The sequences, particularly those of variants, homologues andderivatives of SEQ ID NO: 1, may also have deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent substance. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the secondary bindingactivity of the substance is retained. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine, valine, glycine, alanine, asparagine, glutamine, serine,threonine, phenylalanine, and tyrosine.

The present invention also encompasses conservative substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) that may occur i.e. like-for-like substitution such as basicfor basic, acidic for acidic, polar for polar etc. Non-conservativesubstitution may also occur i.e. from one class of residue to another oralternatively involving the inclusion of unnatural amino acids such asornithine (hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

Conservative substitutions that may be made are, for example within thegroups of basic amino acids (Arginine, Lysine and Histidine), acidicamino acids (glutamic acid and aspartic acid), aliphatic amino acids(Alanine, Valine, Leucine, Isoleucine), polar amino acids (Glutamine,Asparagine, Serine, Threonine), aromatic amino acids (Phenylalanine,Tryptophan and Tyrosine), hydroxyl amino acids (Serine, Threonine),large amino acids (Phenylalanine and Tryptophan) and small amino acids(Glycine, Alanine).

Replacements may also be made by unnatural amino acids include; alpha*and alpha-disubstituted* amino acids, N-alkyl amino acids*, lacticacid*, halide derivatives of natural amino acids such astrifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*,p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyricacid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-aminocaproic acid^(#), 7-amino heptanoic acid*, L-methionine sulfone^(#*),L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*,L-hydroxyproline^(#), L-thioproline*, methyl derivatives ofphenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe(4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionicacid # and L-Phe (4-benzyl)*. The notation * has been utilised for thepurpose of the discussion above (relating to homologous ornon-conservative substitution), to indicate the hydrophobic nature ofthe derivative whereas # has been utilised to indicate the hydrophilicnature of the derivative, #* indicates amphipathic characteristics.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups such as methyl, ethyl or propyl groups inaddition to amino acid spacers such as glycine or β-alanine residues. Afurther form of variation, involves the presence of one or more aminoacid residues in peptoid form, will be well understood by those skilledin the art. For the avoidance of doubt, “the peptoid form” is used torefer to variant amino acid residues wherein the α-carbon substituentgroup is on the residue's nitrogen atom rather than the α-carbon.Processes for preparing peptides in the peptoid form are known in theart, for example Simon R J et al. (1992), Horwell DC. (1995).

In one embodiment, the variant targeting unit used in the homodimericprotein according to the present invention is variant having thesequence of amino acids 5-70 of SEQ ID NO:1 and having at least at least65%, at least 70%, at least 75%, at least 78%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% amino acid sequence identity therewith.

In one aspect, preferably the protein or sequence used in the presentinvention is in a purified form. The term “purified” means that a givencomponent is present at a high level. The component is desirably thepredominant active component present in a composition.

A “variant” or “variants” refers to proteins, polypeptides, units,motifs, domains or nucleic acids. The term “variant” may be usedinterchangeably with the term “mutant.” Variants include insertions,substitutions, transversions, truncations, and/or inversions at one ormore locations in the amino acid or nucleotide sequence, respectively.The phrases “variant polypeptide”, “polypeptide”, “variant” and “variantenzyme” mean a polypeptide/protein that has an amino acid sequence thathas been modified from the amino acid sequence of SEQ ID NO: 1. Thevariant polypeptides include a polypeptide having a certain percent,e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, ofsequence identity with SEQ ID NO: 1, or the amino acid sequence 5-70 ofSEQ ID NO: 1.

“Variant nucleic acids” can include sequences that are complementary tosequences that are capable of hybridizing to the nucleotide sequencespresented herein. For example, a variant sequence is complementary tosequences capable of hybridizing under stringent conditions, e.g., 50°C. and 0.2×SSC (1×SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), tothe nucleotide sequences presented herein. More particularly, the termvariant encompasses sequences that are complementary to sequences thatare capable of hybridizing under highly stringent conditions, e.g., 65°C. and 0.1×SSC, to the nucleotide sequences presented herein. Themelting point (Tm) of a variant nucleic acid may be about 1, 2, or 3° C.lower than the Tm of the wild-type nucleic acid. The variant nucleicacids include a polynucleotide having a certain percent, e.g., 80%, 85%,90%, 95%, or 99%, of sequence identity with the nucleic acid encodingSEQ ID NO: 1 or encoding the monomeric protein which can form thehomodimeric protein according to invention.

The term “homodimeric protein” as used herein refers to a proteincomprising two individual identical strands of amino acids, or subunitsheld together as a single, dimeric protein by either hydrogen bonding,ionic (charged) interactions, actual covalent disulfide bonding, or somecombination of these interactions.

The term “dimerization motif”, as used herein, refers to the sequence ofamino acids between the antigenic unit and the targeting unit comprisingthe hinge region and the optional second domain that may contribute tothe dimerization. This second domain may be an immunoglobulin domain,and optionally the hinge region and the second domain are connectedthrough a linker. Accordingly the dimerization motif serve to connectthe antigenic unit and the targeting unit, but also contain the hingeregion that facilitates the dimerization of the two monomeric proteinsinto a homodimeric protein according to the invention.

The term “targeting unit” as used herein refers to a unit that deliversthe protein with its antigen to mouse or human APC for MHC classII-restricted presentation to CD4+ T cells or for providing crosspresentation to CD8+ T cells by MHC class I restriction.

The term “antigenic unit” as used herein refers to any molecule, such asa peptide which is able to be specifically recognized by an antibody orother component of the immune system, such as a surface receptor onT-cells. Included within this definition are also immunogens that areable to induce an immune response, such as idiotype immunogens ofantibodies. The terms “epitope” or “antigenic epitope” is used to referto a distinct molecular surface, such as a molecular surface provided bya short peptide sequence within an antigenic unit. In some embodimentsthe antigenic unit comprises two ore more antigenic epitopes.

The term “hinge region” refers to a peptide sequence of the homodimericprotein that facilitates the dimerization, such as through the formationof an interchain covalent bond(s), e.g. disulfide bridge(s). The hingeregion may be Ig derived, such as hinge exons h1+h4 of an Ig, such asIgG3.

The term “immunostimulating composition” as used herein refers to anytherapeutic composition that is capable to activate the immune system,e.g., by activating or inhibiting lymphocyte functions, in particularT-cell functions like T-cell activation.

SPECIFIC EMBODIMENTS OF THE INVENTION

In some embodiments the antigenic unit is a cancer associated or acancer specific antigen.

The term “cancer associated antigen” refers to any antigen, which in notnecessarily specific for a certain cancer, but overexpressed on thesurface of the cancer cells of this cancer. The term may be usedinterchangeably with the term “cancer antigens”.

In some embodiments the antigenic unit is an antigenic scFv. In someembodiments a linker, such as a (G₄S)₃ linker, connects the V_(H) andV_(L) in the antigenic scFv. In some embodiments the antigenic scFv isderived from a monoclonal Ig produced by myeloma or lymphoma cells.

In some embodiments the antigenic unit is a telomerase, or a functionalpart thereof. In some embodiments the telomerase is hTERT.

In some embodiments the antigenic unit is a melanoma antigen. In someembodiments the melanoma antigen is tyrosinase, TRP-1, or TRP-2.

In some embodiments the antigenic unit is a prostate cancer antigen. Insome embodiments the prostate cancer antigen is PSA.

In some embodiments the antigenic unit is a cervix cancer antigen. Insome embodiments the cervix cancer antigen is selected from the listconsisting of human papilloma virus E1, E2, E4, E6, and E7.

In some embodiments the antigenic unit is derived from a bacterium.

In some embodiments the bacterium derived antigenic unit is atuberculosis antigen.

In some embodiments the bacterium derived antigenic unit is abrucellosis antigen.

In some embodiments the antigenic unit is derived from a virus.

In some embodiments the virus derived antigenic unit is derived fromHIV. In some embodiments the HIV derived antigenic unit is derived fromgp120 or Gag.

In some embodiments the antigenic unit is selected from the listconsisting of influenza virus hemagglutinin (HA), nucleoprotein, and M2antigen; Herpes simplex 2 antigen glycoprotein D; and a Human Papillomavirus antigen, such as any one selected from the list consisting of E1,E2, E6, E7, L1 and L2.

In some embodiments the dimerization motif comprises a hinge region andoptionally another domain that facilitate dimerization, such as animmunoglobulin domain, optionally connected through a linker.

In some embodiments the hinge region is Ig derived, such as derived fromIgG3.

In some embodiments the hinge region has the ability to form one, two,or several covalent bonds. In some embodiments the covalent bond is adisulphide bridge.

In some embodiments the immunoglobulin domain of the dimerization motifis a carboxyterminal C domain, or a sequence that is substantiallyhomologous to said C domain.

In some embodiments the carboxyterminal C domain is derived from IgG.

In some embodiments the immunoglobulin domain of the dimerization motifhas the ability to homodimerize.

In some embodiments the immunoglobulin domain of the dimerization motifhas the ability to homodimerize via noncovalent interactions. In someembodiments the noncovalent interactions are hydrophobic interactions.

In some embodiments the dimerization domain does not comprise the CH2domain.

In some embodiments the dimerization motif consist of hinge exons h1 andh4 connected through a linker to a C_(H)3 domain of human IgG3.

In some embodiments the linker that connect the hinge region and anotherdomain that facilitate dimerization, such as an immunoglobulin domain,is a G₃S₂G₃SG linker.

In some embodiments the antigenic unit and the dimerization motif isconnected through a linker, such as a GLSGL linker.

In some embodiments the targeting unit comprises amino acids 3-70 of SEQID NO: 1.

In some embodiments the targeting unit consists of amino acids 5-70 ofSEQ ID NO: 1.

In some embodiments the targeting unit consists of amino acids 3-70 ofSEQ ID NO: 1.

In some embodiments the targeting unit consists of amino acids 1-70 ofSEQ ID NO: 1.

In some embodiments the homodimeric protein do not comprise amino acids1-70 of SEQ ID NO:1.

In some embodiments the targeting unit comprises amino acids 3-70 of SEQID NO:2.

In some embodiments the targeting unit consists of amino acids 5-70 ofSEQ ID NO:2.

In some embodiments the targeting unit consists of amino acids 3-70 ofSEQ ID NO:2.

In some embodiments the targeting unit consists of amino acids 1-70 ofSEQ ID NO:2.

In some embodiments the homodimeric protein do not comprise amino acids1-70 of SEQ ID NO:2.

In some embodiments the targeting unit consists of not more than 68amino acids, such as 68, 67, or 66 amino acids.

In some embodiments the targeting unit do not contain the amino acidsequence AP at positions 1 and 2 of the targeting unit.

In some embodiments the homodimeric protein is a first homodimericprotein having increased affinity as compared to the affinity of asecond homodimeric protein, which second homodimeric protein onlydiffers from said first homodimeric protein by having a targeting unit,which consists of amino acids 1-70 of SEQ ID NO:2; the increasedaffinity being for any one chemokine receptor selected from CCR1, CCR3and CCR5. In some embodiments the nucleic acid molecule according toinvention is comprised by a vector.

In some embodiments the nucleic acid molecule according to the inventionis formulated for administration to a patient to induce production ofthe homodimeric protein in said patient.

In some embodiments the vaccine or immunostimulating compositionaccording to the invention comprises a pharmaceutically acceptablecarrier and/or adjuvant.

In some embodiments the cancer treated by a vaccine or immunostimulatingcomposition or pharmaceutical compositions according to the presentinvention is multiple myeloma or lymphoma, malignant melanoma, HPVinduced cancers, prostate cancer, breast cancer, lung cancer, ovariancancer, and/or liver cancer.

In some embodiments the infectious disease treated by a vaccine orimmunostimulating composition or pharmaceutical compositions accordingto the present invention is selected from the list consisting ofinfluenza, Herpes, CMV, HPV, HBV, brucellosis, HIV, HSV-2 andtuberculosis.

NUMBERED EMBODIMENTS OF THE INVENTION

1. A homodimeric protein of two identical amino acid chains, each aminoacid chain comprising a targeting unit comprising an amino acid sequencehaving at least 80% sequence identity to the amino acid sequence 5-70 ofSEQ ID NO: 1, and an antigenic unit, the targeting unit and theantigenic unit being connected through a dimerization motif.

2. The homodimeric protein according to embodiment 1, wherein theantigenic unit is an antigenic scFv.

3. The homodimeric protein according to embodiments 1 or 2, wherein alinker, such as a (G₄S)₃ linker, connects the V_(H) and V_(L) in theantigenic scFv.

4. The homodimeric protein according to any one of embodiments 1-3,wherein the antigenic scFv is derived from a monoclonal Ig produced bymyeloma or lymphoma cells.

5. The homodimeric protein according to embodiment 1, wherein theantigenic unit is a telomerase, or a functional part thereof.

6. The homodimeric protein according to embodiment 5, wherein saidtelomerase is hTERT.

7. The homodimeric protein according to embodiment 1, wherein theantigenic unit is derived from a bacterium.

8. The homodimeric protein according to embodiment 7, wherein thebacterium derived antigenic unit is selected from a tuberculosis antigenand a brucellosis antigen.

9. The homodimeric protein according to embodiment 1, wherein theantigenic unit is derived from a virus.

10. The homodimeric protein according to embodiment 9, wherein the virusderived antigenic unit is derived from HIV.

11. The homodimeric protein according to embodiment 10, wherein the HIVderived antigenic unit is derived from gp120 or Gag.

12. The homodimeric protein according to embodiment 9, wherein theantigenic unit is selected from the list consisting of influenza virushemagglutinin (HA), nucleoprotein, and M2 antigen; and Herpes simplex 2antigen glycoprotein D.

13. The homodimeric protein according to embodiment 1, wherein theantigenic unit is a cancer associated or a cancer specific antigen.

14. The homodimeric protein according to embodiment 13, wherein thecancer antigenic unit is a melanoma antigen, such as the melanomaantigens tyrosinase, TRP-1 or TRP2.

15. The homodimeric protein according to embodiment 13, wherein thecancer antigenic unit is a prostate cancer antigen, such as the prostatecancer antigen PSA.

16. The homodimeric protein according to embodiment 13, wherein thecancer antigenic unit is a cervix cancer antigen, such as the cervixcancer antigen selected from the list consisting of E1, E2, E4, E6, E7,L1 and L2.

17. The homodimeric protein according to any one of embodiments 1-16,wherein the dimerization motif comprises a hinge region and optionallyanother domain that facilitate dimerization, such as an immunoglobulindomain, optionally connected through a linker.

18. The homodimeric protein according to embodiment 17, wherein thehinge region is Ig derived, such as derived from IgG3.

19. The homodimeric protein according to any one of embodiments 17-18,wherein the hinge region has the ability to form one, two, or severalcovalent bonds.

20. The homodimeric protein according to any one of embodiments 17-19,wherein the covalent bond is a disulphide bridge.

21. The homodimeric protein according to any one of embodiments 17-20,wherein the immunoglobulin domain of the dimerization motif is acarboxyterminal C domain, or a sequence that is substantially homologousto said C domain.

22. The homodimeric protein according to embodiment 21, wherein thecarboxyterminal C domain is derived from IgG.

23. The homodimeric protein according to any one of embodiments 17-22,wherein the immunoglobulin domain of the dimerization motif has theability to homodimerize.

24. The homodimeric protein according to any one of embodiments 17-23,wherein said immunoglobulin domain has the ability to homodimerize vianoncovalent interactions.

25. The homodimeric protein according to embodiment 24, wherein saidnoncovalent interactions are hydrophobic interactions.

26. The homodimeric protein according to any one of embodiments 1-25,wherein said dimerization domain does not comprise the CH2 domain.

27. The homodimeric protein according to any one of embodiments 1-26,wherein the dimerization motif consist of hinge exons h1 and h4connected through a linker to a C_(H)3 domain of human IgG3.

28. The homodimeric protein according to any one of embodiments 17-27,wherein said linker is a G₃S₂G₃SG linker.

29. The homodimeric protein according to any one of embodiments 1-28,wherein said antigenic unit and the dimerization motif is connectedthrough a linker, such as a GLSGL linker.

30. The homodimeric protein according to any one of embodiments 1-29,wherein said targeting unit comprises amino acids 3-70 of SEQ ID NO: 1.

31. The homodimeric protein according to any one of embodiments 1-29,wherein said targeting unit consist of amino acids 5-70 of SEQ ID NO: 1.

32. The homodimeric protein according to any one of embodiments 1-29,wherein said targeting unit consist of amino acids 3-70 of SEQ ID NO: 1.

33. The homodimeric protein according to any one of embodiments 1-30,wherein said targeting unit consist of amino acids 1-70 of SEQ ID NO: 1.

34. The homodimeric protein according to any one of embodiments 1-29,which homodimeric protein do not comprise amino acids 1-70 of SEQ ID NO:1.

35. The homodimeric protein according to any one of embodiments 1-29,wherein said targeting unit comprises amino acids 3-70 of SEQ ID NO:2.

36. The homodimeric protein according to any one of embodiments 1-29,wherein said targeting unit consist of amino acids 5-70 of SEQ ID NO:2.

37. The homodimeric protein according to any one of embodiments 1-29,wherein said targeting unit consist of amino acids 3-70 of SEQ ID NO:2.

38. The homodimeric protein according to any one of embodiments 1-29,wherein said targeting unit consist of amino acids 1-70 of SEQ ID NO:2.

39. The homodimeric protein according to any one of embodiments 1-29,which homodimeric protein do not comprise amino acids 1-70 of SEQ IDNO:2.

40. The homodimeric protein according to any one of embodiments 1-32,35-37, wherein each said targeting unit consist of not more than 68amino acids, such as 68, 67, or 66 amino acids.

41. The homodimeric protein according to any one of embodiments 1-30,35, wherein said targeting unit do not contain the amino acid sequenceAP at positions 1 and 2 of the targeting unit.

42. The homodimeric protein according to any one of embodiments 1-41,which homodimeric protein have increased affinity for any one chemokinereceptor selected from CCR1, CCR3 and CCR5 as compared to the affinityof the same homodimeric protein, wherein the targeting unit consists ofamino acids 1-70 of SEQ ID NO:2.

43. A nucleic acid molecule encoding the monomeric protein which canform the homodimeric protein according to any one of embodiments 1-42.

44. The nucleic acid molecule according to embodiment 43 comprised by avector.

45. The nucleic acid molecule according to embodiments 43 or 44formulated for administration to a patient to induce production of thehomodimeric protein in said patient.

46. The homodimeric protein according to any one of embodiments 1-42 orthe nucleic acid molecule according to embodiments 43 or 44 for use as amedicament.

47. A pharmaceutical composition comprising the homodimeric proteinaccording to any one of embodiments 1-42, or the nucleic acid moleculeaccording to embodiments 43 or 44.

48. A host cell comprising the nucleic acid molecule according toembodiments 43 or 44.

49. A method for preparing a homodimeric protein according to any one ofembodiments 1-42, the method comprising

-   -   a) transfecting the nucleic acid molecule according to        embodiments 43 or 44 into a cell population;    -   b) culturing the cell population;    -   c) collecting and purifying the homodimeric protein expressed        from the cell population.

50. A vaccine against a cancer or an infectious disease comprising animmunologically effective amount of a homodimeric protein according toany one of embodiments 1-42 or nucleic acid molecule according toembodiments 43 or 44 encoding the monomeric protein which can form thehomodimeric protein, wherein said vaccine is able to trigger both aT-cell- and B-cell immune response and wherein said homodimeric proteincontain an antigenic unit specific for said cancer or infectiousdisease.

51. The vaccine according to embodiment 50 further comprising apharmaceutically acceptable carrier and/or adjuvant.

52. The vaccine according to embodiments 50 or 51, wherein said canceris multiple myeloma or lymphoma, malignant melanoma, HPV inducedcancers, prostate cancer, breast cancer, lung cancer, ovarian cancer,and/or liver cancer.

53. The vaccine according to embodiment 50 or 51, wherein saidinfectious disease is selected from the list consisting of tuberculosis,Influenza, Herpes, CMV, HPV, HBV, HIV, brucellosis, and/or HSV-2.

54. A method of treating a cancer or an infectious disease in a patient,the method comprising administering to the patient in need thereof, ahomodimeric protein according to any one of embodiments 1-42, or thenucleic acid molecule according to embodiments 43 or 44 encoding themonomeric protein which can form the homodimeric protein, wherein saidhomodimeric protein contain an antigen unit specific for said cancer orinfectious disease.

Example 1

Mice and Cell Lines

BALB/c mice were obtained from Taconic (Ry, Denmark). Id(λ2³¹⁵)-specificT-cell receptor (TCR) transgenic mice have been described (see Bogen Bet al. Eur J Immunol 1992 March; 22(3):703-9 and Snodgrass H R et al.Eur J Immunol 1992 August; 22(8):2169-72). The TCR recognizes aa 91-101of the λ2³¹⁵ light chain, produced by the IgA MOPC315 mouseplasmacytoma, presented on the I-E^(d) class II molecules. The studieswere approved by the National Committee for Animal Experiments (Oslo,Norway). MOPC 315.4 (IgA, λ2³¹⁵), HEK 293 and HEK 293E cells were fromATCC. HEK 293 stably transfected with hCCR5 and hCCR1 were kindlyprovided by Mario Mellado (Madrid, Spain) and Zack Howard (Frederick,Md.), respectively. HEK 293 stably transfected with Rhesus macaque(GenBank AF005660) were obtained from Pfizer Inc., (Groton, Conn.). Themurine lymphoma Esb/MP cells were kindly provided by Jo Van Damme(Leuven, Belgium).

Cloning of Human MIP1a/CCL3 (LD78α or LD78β-Encoding Vaccibodies) Genesencoding for mature LD78α and LD78β (GenBank NM_002983 and NG_004113,respectively) were amplified from cDNA of CD14-enriched, bonemarrow-derived monocytes from a healthy donor. Forward primers (BsmIrestriction site, in italic) were:

LD78α: (SEQ ID NO: 3) GGTGTGCATTCCGCATCACTTGCTGCTGAC; LD78β:(SEQ ID NO: 4) GGTGTGCATTCCGCACCACTTGCTGCTGAC;and reverse primer (BsiWI restriction site, in italic) wasGACGTACGACTCACCTGCAACTCAGCTCCAGGTC (SEQ ID NO:5). The 68 aa. long (3-70)LD78β-2 was cloned using forward primer (BsmI restriction site, initalic): GGTGTGCATTCCCTTGCTGCTGACACGCC (SEQ ID NO:6).

Point mutated LD78α and LD78β carrying an S instead of a C residue atposition 11 were generated by quick change PCR using the followingprimers: forward CCGACCGCCTCCTGCTTCAG (SEQ ID NO:7) and reverseCTGAAGCAGGAGGCGGTCGG (SEQ ID NO:8). The amplified chemokine genes wereinserted into the targeting cassette of vaccibody construct IlhFpLNOH2(see Fredriksen A B et al. Mol Ther 2006 April; 13(4):776-85) by use ofBsmI/BsiWI restriction sites. The resulting vaccibody construct encodedfor homodimeric proteins with hCCL3-derived targeting units and MOPC315scFv in a VH-VL orientation as antigenic unit, connected via ahomodimerizing motif consisting of human hinge exons h1 and h4 and CH3domain of IgG3.

The antigenic unit (scFv³¹⁵) in vaccibodies described above wereexchanged with either paired murine Cκ domains (Tunheim G et al. Vaccine2007 Jun. 11; 25(24):4723-34) or influenza virus hemagglutinin (HA) fromH1N1 A/Puerto Rico/8/34 (Mt. Sinai) (G. Grødeland, manuscript inpreparation).

Assessment of Vaccine Protein Production

Supernatants of transiently transfected 293E cells were tested in thefollowing ELISAs. scFv³¹⁵ vaccibodies: DNP-BSA (binds to M315) as coatand biotinylated monoclonal HP6017 (anti-CH3 dimerization motif) fordetection; HA vaccibodies: MCA878G (anti-CH3 dimerization motif) as coatand anti-HA biotinylated mAB H36-4-52 for detection; mouse CκCκvaccibodies: 187.1 mAb (binds to mouse Cκ) as coat and biotinylated187.1 for detection.

Production, Purification, Quantitation and Proteomic Characterization ofVaccibody Proteins

Vaccibody proteins having scFv³¹⁵ as antigenic unit were affinitypurified from supernatants of stably transfected NS0 cells on DNP (boundby M315) Sepharose columns. Purified proteins were loaded onto a 4-20%Tris-glycine gel. Following membrane transfer, proteins were detectedwith either biotinylated HP6017 or Ab2-1.4 (binds to M315) mAbs followedby streptavidin HRP. Vaccibody proteins were quantified by Bradford andELISA using BSA and mCCL3 vaccibody (see Fredriksen A B et al. Mol Ther2006 April; 13(4):776-85) as standards, respectively. Protein bandscorresponding to LD78β and LD78β-2 vaccibodies with Fv³¹⁵ were excisedfrom a Coomassie-stained polyacrylamide gel and subjected to trypticin-gel digestion as previously described.

Binding to Human and Murine CCR5 and CCR1

Vaccibody proteins at concentrations ranging from 0.2 to 25 μg/mL wereused to stain parental or stably transfected HEK 293 cells or BALB/csplenocytes (gated by FSC/SSC and on CD11b+CD3− cells). Bound vaccibodyproteins were detected by biotinylated HP6017 (binds to CH3 of hIgG3) orAb2-1.4 (binds to M315) mAbs followed by streptavidin PE. Cells wereanalyzed on a FACScalibur.

Chemotaxis Assay

Cell migration in vitro was assessed by a 24-well transwell plate(Corning) as previously described. Either 8 μm or 5 μm porepolycarbonate membranes were used for HEK 293 cells and Esb/MP,respectively. Recombinant chemokines were from Peprotech. The results(mean±SE of duplicate samples) are presented as chemotactic index,defined as the fold increase in the number of migrating cells in thepresence of chemotactic factors over the spontaneous cell migration(i.e., in the presence of medium alone).

T Cell Stimulation Assays

BALB/c splenocytes were irradiated (8 Gy) and mixed with vaccibodyproteins containing scFv³¹⁵ at concentration ranging from 20 to 0.04μg/mL before addition of in vitro polarized Id³¹⁵-specific Th2 cellsderived from TCR-transgenic mice. An Id peptide corresponding tosequence 89-107 of λ2³¹⁵ was used as a positive control.

Human PBMC from three different DR4*01 healthy donors were mixed withsupernatants from transiently transfected 293E cells, containingvaccibody proteins with mouse CκCκ as antigenic unit, before irradiation(20 Gy) and addition of T18 T cell clone that recognizes aa. 40-48 ofmurine Ck presented by DR4*01. After 48 hrs plates were pulsed with³H-thymidine for 24 hrs before harvesting.

Mouse Immunizations

Vaccibody DNA was purified using Endofree-mega plasmid purificationsystem (Qiagen). 25 μL solution of 0.5 mg/mL vaccibody DNA in sterile0.9% NaCl was injected intradermally in the lower back of mice, on bothsides, followed by electroporation using Dermavax (Cytopulse, Sweden).Groups consisted of 3 to 7 mice.

Anti-Id315 Antibodies Measurement

Mice were bled three, four and six weeks following a singleimmunization. Myeloma protein M315 (IgA, A2) was used as coat andanti-Id³¹⁵ Abs in mouse sera were detected by biotinylated anti-mouseIgG1^(a) or anti-mouse IgG2a^(a) (BD Pharmingen). Endpoint titers werecalculated.

Elispot Assay

Millipore Multiscreen plates (Millipore, Billerca, Mass., USA) werecoated with anti-mouse IFNγ (AN18) (12 μg/ml) and then blocked for 2 hwith RPMI-1640 (Invitrogen, NY, USA) containing 10% FCS. Single cellsplenocytes were prepared individually from mice DNA-vaccinated 3 weeksearlier with HA-containing vaccibodies or NaCl, and incubated overnightat 10⁶, 5×10⁵ and 2.5×10⁵ cells/well with one of the followingHA-derived peptides from ProImmune (Oxford, UK): SVSSFERFEIPK (aa.107-119, I-E^(d)-restricted), HNTNGVTAACSHEG (aa. 126-138,I-A^(d)-restricted) or IYSTVASSL (aa. 633-641, K^(d) restricted). Plateswere washed in PBS and adherent cells lysed by a five minute incubationin de-ionized water prior to incubation with biotinylated anti-mouseIFNγ (1 μg/ml) (XMG1.2, Pharmingen) and Streptavidine alkalinephosphatase conjugate (1:3000) (GE Healthcare, Little ChalfontBuckinghamshire, UK). IFNγ-producing cells were detected by using theBCIP/NBT kit (Zymed Laboratories Inc, Carlsbad, Calif., USA), andcounting was performed with KS EliSpot version 4.3.56 from Zeiss on aZeiss Axioplan 2 imaging system (objective: Epiplan-Neofluar 5x,442320).

Vaccination of Mice with Vaccibody-HA Constructs.

Mice were anesthetized, shaved, and vaccinated intradermally with 25 μlDNA (0.5 mg/ml) on each side of the lower back region immediatelyfollowed by skin electroporation (DermaVax/CytoPulse). 14 days later,the mice were anesthetized with hypnorm/dormicum and challenged with 20μl influenza (λ/Puerto Rico/8/34 (Mt. Sinai) virus (5×LD50). Followingchallenge, the mice were weighed and closely monitored for clinicalsigns.

Construction and Expression of Human CCL3-Based Vaccibodies

Homodimeric vaccibodies were constructed that have various hCCL3-basedtargeting units, a human Ig-derived homodimerization unit and variousantigenic units, as indicated in FIG. 1A. The NH₂ terminal aa. sequenceof the employed LD78α, LD78β, LD78β-2 and the putative effect of theC11S point mutation on chemokine structure LD78β(C11S) are shown inFIGS. 1B and 1C, respectively. All vaccibodies were expressed atcomparable levels by transiently transfected 293E cells, except LD78β-2vaccibodies which were consistently expressed to a lower extent (FIG. 2A-C).

The integrity of the vaccibody proteins having scFv³¹⁵ as antigenic unitwas tested by SDS-PAGE, where single bands of about 110 kDa were visiblethat, following membrane blotting, could be stained with appropriatemAbs (FIG. 2 D and data not shown for other constructs). The apparentlyincreased size of the mutated (C11S) vaccibody is likely due to anincrease in Stokes radius due to abrogation of a S—S bond (FIG. 1C).Under reducing conditions, single bands of about 55 kDA, correspondingto monomers were observed, as would be expected (data not shown).

The NH₂ terminal aa. sequence of LD78β vaccibody proteins was furtherascertained since NS0 cells or fetal bovine serum in culture mediumcould have CD26 activity resulting in posttranslational modification ofLD78β. Analysis of tryptic digests of LD78βIhF and LD78β-2IhF vaccibodyproteins by MALDI-TOF mass spectrometry showed exclusively signals atm/z 1988.89 and m/z 1820.78, corresponding to the N-terminal peptidesAPLAADTPTACCFSYTSR (SEQ ID NO:9) and LAADTPTACCFSYTSR (SEQ ID NO: 10),respectively (data not shown). Thus, pure full length LD78β orNH₂-truncated LD78β-2 vaccibodies can be expressed and purified fromstably transfected NS0 cells.

LD78β Vaccibodies and NH₂-Truncated Version Bind Human and MurineChemokine Receptors

Given the variability in CCR5 expression between individuals, HEK 293stably transfected with hCCR5, rather than PBMC, were used forfunctional studies. LD78β and LD78β-2 vaccibody, but not their pointmutated counterpart, bound hCCR5-transfected HEK 293 cells (FIG. 3 A, Band data not shown). NH₂-truncated LD78β-2 vaccibody displayed strongerbinding than full length LD78β vaccibody. Point mutated LD78β (C11S)vaccibody did not bind hCCR5-transfected cells (FIG. 3A). LD78β-2, butnot LD78β vaccibodies, also bound hCCR1-expressing HEK 293 (FIG. 3 C, Dand data not shown), which is in agreement with previous reports. Therewas no staining of parental, untransfected HEK 293 cells (not shown).

LD78β vaccibody, but not its point mutated counterpart, bound CD11b+BALB/c splenocytes (FIG. 4 A), and induced chemotaxis of Esb/MP cells(FIG. 4 B), thus providing the rationale for testing LD78β-expressingvaccibodies in mice.

Delivery of Antigen to APC Via Chemokine Receptors Improves T-CellResponses In Vitro in Mouse and Human Systems

BALB/c splenocytes mixed with LD78β vaccibodies that express scFv³¹⁵antigenic unit induced proliferation of Id³¹⁵-specific Th2 cells fromTCR transgenic mice (FIG. 5 A). Similar vaccibodies where the C11Smutation had been introduced had a ˜100 fold decreased ability tostimulate T cells. When comparing equimolar concentrations of LD78βVaccibody and an Id peptide encompassing the CDR3 mutations, theVaccibody was found to be up to 30 times more effective than the peptideat loading the APCs for antigen presentation (not shown).

As for human T cell responses, LD78β vaccibodies that express murineCκCκ as a model antigen were mixed with donor PBMCs and cloned T18 CD4+T cells that recognize aa 40-48 of murine Cκ in the context of DR4*01.The difference between wild type and point mutated LD78β was lesspronounced than in the mouse system (FIG. 5 B). Furthermore, targetedantigen delivery is demonstrated by superiority of LD78β overnon-targeted NIP-specific vaccibody (FIG. 5B). Importantly, LD78β-2vaccibodies outperformed LD78β vaccibodies. Similar results wereobtained using three different donors (not shown).

Improved Anti-Id Humoral Response Induced in Mice by LD78β VaccibodiesContaining scFv³¹⁵

The V_(H)+V_(L) Id of M315 myeloma protein is a very weak self antigen,in fact an extensive immunization scheme including multipleimmunizations with complete and incomplete Freund's adjuvant wasrequired to detect anti-Id antibodies. We therefore tested if miceinjected intradermally with LD78βscFv315 vaccibody DNA, combined withelectroporation, developed anti-Id antibodies. Anti-Id³¹⁵ IgG1 (FIG. 6A)and IgG2a (FIG. 6B) responses were detected in mice that had beenimmunized with LD78β-encoding vaccibodies, further demonstrating thatconformational integrity of scFv³¹⁵ is maintained in LD78β vaccibody(FIG. 6 ). IgG1 responses were recorded to a significantly lesser extentin mice receiving the C11S point-mutated vaccibody, whereas thedifference for IgG2a was not significant. Furthermore, statisticallysignificant lower Ab responses were observed for both IgG1 and IgG2a inmice that had been immunized with non targeted control vaccibodies(anti-NIP). These result overall suggest that targeted antigen deliveryimproves antibody responses to a weak self model tumor antigen.

Induction of Influenza Hemagglutinin-Specific CD4+ and CD8+ T-CellResponses in Mice Following Vaccibody Administration

Induction of CD8+ T cell responses was investigated in an influenzamodel where hemagglutinin (HA) served as the target antigen. HA fromstrain A/Puerto Rico/8/34 (Mt. Sinai) (H1N1) is known to express threeepitopes to which BALB/c mice (H-2^(d)) respond. Two of these are MHCclass II-restricted, SVSSFERFEIPK (SEQ ID NO:11) (aa. 107-119)restricted by I-E^(d) and HNTNGVTAACSHEG (SEQ ID NO:12) (aa. 126-138)restricted by I-A^(d), respectively. The third epitope, IYSTVASSL (SEQID NO:13) (aa. 633-641), is MHC class I-restricted (K^(d)). Following asingle intradermal LD78β vaccibody DNA immunization and electroporation,significantly increased IFNγ responses to the class I epitope wereobserved for targeted vs. non-targeted (C11S) vaccibodies and shamimmunization (NaCl) (FIG. 7 A). Responses to class II epitopes wereslightly elevated but the effect of targeting was not significant (onlyone immunization was delivered in the present experiments) (FIG. 7 A).

LD78β Vaccibody Binds to Rhesus Macaque CCR5

CCR5 is conserved across different species, including monkey. Human andmacaque CCR5 genes have very close aa. homology (98%). Like humans,macaques have two CCL3 isoforms. LD78β and LD78β-2 vaccibodies bound ina dose-dependent fashion Rhesus macaque CCR5-expressing HEK 293 cells,whereas the C11S point mutated LD78β did not bind the same cells (FIG. 8, and data not shown). This result indicates that vaccibodies with LD78βand LD78β-2 intended for human use not only can be tested in mice, asabove, but also in Rhesus macaques, prior to any human application.

LD78β-HA Vaccibody but not LD78β-2-HA Vaccibody Protects Mice fromInfluenza.

As shown in FIG. 9 , mice were vaccinated with either of the two formsof LD78β, LD78β or LD78β-2. The full length version of LD78β was shownto have superior effect in terms of protecting mice from influenzainfection.

Sequences:

LD78β (SEQ ID NO: 1):APLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVC ADPSEEWVQKYVSDLELSALD78α (SEQ ID NO: 2):ASLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPGVIFLTKRSRQVC ADPSEEWVQKYVSDLELSA

Items

1. A homodimeric protein of two identical amino acid chains, each aminoacid chain comprising a targeting unit comprising an amino acid sequencehaving at least 80% sequence identity to the amino acid sequence 5-70 ofSEQ ID NO: 1, and an antigenic unit, the targeting unit and theantigenic unit being connected through a dimerization motif.

2. The homodimeric protein according to item 1, wherein said targetingunit comprises amino acids 3-70 of SEQ ID NO: 1.

3. The homodimeric protein according to items 1 or 2, wherein saidtargeting unit consist of amino acids 5-70 of SEQ ID NO: 1.

4. The homodimeric protein according to any one of items 1-3, whereinsaid targeting unit consist of amino acids 3-70 of SEQ ID NO: 1.

5. The homodimeric protein according to any one of items 1-4, whereinsaid targeting unit consist of amino acids 1-70 of SEQ ID NO: 1.

6. The homodimeric protein according to any one of items 1-5, whereinthe antigenic unit is an antigenic scFv.

7. The homodimeric protein according to any one of items 1-6, wherein alinker, such as a (G₄S)₃ linker, connects the V_(H) and V_(L) in theantigenic scFv.

8. The homodimeric protein according to any one of items 1-7, whereinthe antigenic scFv is derived from a monoclonal Ig produced by myelomaor lymphoma cells.

9. The homodimeric protein according to any one of items 1-5, whereinthe antigenic unit is a telomerase, or a functional part thereof.

10. The homodimeric protein according to item 9, wherein said telomeraseis hTERT.

11. The homodimeric protein according to any one of item 1-5, whereinthe antigenic unit is derived from a bacterium.

12. The homodimeric protein according to item 11, wherein the bacteriumderived antigenic unit is selected from a tuberculosis antigen and abrucellosis antigen.

13. The homodimeric protein according to any one of items 1-5, whereinthe antigenic unit is derived from a virus.

14. The homodimeric protein according to item 13, wherein the virusderived antigenic unit is derived from HIV.

15. The homodimeric protein according to item 14, wherein the HIVderived antigenic unit is derived from gp120 or Gag.

16. The homodimeric protein according to item 13, wherein the antigenicunit is selected from the list consisting of influenza virushemagglutinin (HA), nucleoprotein, M2 antigen; Herpes simplex 2 antigenglycoprotein D; and a Human Papilloma virus antigen, such as any oneselected from the list consisting of E1, E2, E6, E7, L1 and L2.

17. The homodimeric protein according to any one of items 1-5, whereinthe antigenic unit is a cancer associated or a cancer specific antigen.

18. The homodimeric protein according to item 17, wherein the cancerantigenic unit is a melanoma antigen, such as the melanoma antigenstyrosinase, TRP-1 or TRP2.

19. The homodimeric protein according to item 17, wherein the cancerantigenic unit is a prostate cancer antigen, such as the prostate cancerantigen PSA.

20. The homodimeric protein according to item 17, wherein the cancerantigenic unit is from human papilloma virus, such as the cervix cancerantigen selected from the list consisting of E1, E2, E4, E6 and E7.

21. The homodimeric protein according to any one of items 1-20, whereinthe dimerization motif comprises a hinge region and optionally anotherdomain that facilitate dimerization, such as an immunoglobulin domain,optionally connected through a linker.

22. The homodimeric protein according to item 21, wherein the hingeregion is Ig derived, such as derived from IgG3.

23. The homodimeric protein according to any one of items 21-22, whereinthe hinge region has the ability to form one, two, or several covalentbonds.

24. The homodimeric protein according to any one of items 21-23, whereinthe covalent bond is a disulphide bridge.

25. The homodimeric protein according to any one of items 21-24, whereinthe immunoglobulin domain of the dimerization motif is a carboxyterminalC domain, or a sequence that is substantially homologous to said Cdomain.

26. The homodimeric protein according to item 25, wherein thecarboxyterminal C domain is derived from IgG.

27. The homodimeric protein according to any one of items 21-26, whereinthe immunoglobulin domain of the dimerization motif has the ability tohomodimerize.

28. The homodimeric protein according to any one of items 21-27, whereinsaid immunoglobulin domain has the ability to homodimerize vianoncovalent interactions.

29. The homodimeric protein according to item 28, wherein saidnoncovalent interactions are hydrophobic interactions.

30. The homodimeric protein according to any one of items 1-29, whereinsaid dimerization domain does not comprise the CH2 domain.

31. The homodimeric protein according to any one of items 1-30, whereinthe dimerization motif consist of hinge exons h1 and h4 connectedthrough a linker to a C_(H3) domain of human IgG3.

32. The homodimeric protein according to any one of items 21-31, whereinsaid linker is a G₃S₂G₃SG linker.

33. The homodimeric protein according to any one of items 1-32, whereinsaid antigenic unit and the dimerization motif is connected through alinker, such as a GLSGL linker.

34. The homodimeric protein according to any one of items 1-33, whichhomodimeric protein do not comprise amino acids 1-70 of SEQ ID NO:1.

35. The homodimeric protein according to any one of items 1-33, whereinsaid targeting unit comprises amino acids 3-70 of SEQ ID NO:2.

36. The homodimeric protein according to any one of items 1-33, whereinsaid targeting unit consist of amino acids 5-70 of SEQ ID NO:2.

37. The homodimeric protein according to any one of items 1-33, whereinsaid targeting unit consist of amino acids 3-70 of SEQ ID NO:2.

38. The homodimeric protein according to any one of items 1-33, whereinsaid targeting unit consist of amino acids 1-70 of SEQ ID NO:2.

39. The homodimeric protein according to any one of items 1-33, whichhomodimeric protein do not comprise amino acids 1-70 of SEQ ID NO:2.

40. The homodimeric protein according to any one of items 1-4, 6-37, 39,wherein each said targeting unit consist of not more than 68 aminoacids, such as 68, 67, or 66 amino acids.

41. The homodimeric protein according to any one of items 1-4, 6-37,39-40, wherein said targeting unit do not contain the amino acidsequence AP at positions 1 and 2 of the targeting unit.

42. The homodimeric protein according to any one of items 1-41, which isa first homodimeric protein having increased affinity as compared to theaffinity of a second homodimeric protein, which second homodimericprotein only differs from said first homodimeric protein by having atargeting unit, which consists of amino acids 1-70 of SEQ ID NO:2; theincreased affinity being for any one chemokine receptor selected fromCCR1, CCR3 and CCR5.

43. A nucleic acid molecule encoding the monomeric protein which canform the homodimeric protein according to any one of items 1-42.

44. The nucleic acid molecule according to item 43 comprised by avector.

45. The nucleic acid molecule according to items 43 or 44 formulated foradministration to a patient to induce production of the homodimericprotein in said patient.

46. The homodimeric protein according to any one of items 1-42 or thenucleic acid molecule according to items 43 or 44 for use as amedicament.

47. A pharmaceutical composition comprising the homodimeric proteinaccording to any one of items 1-42, or the nucleic acid moleculeaccording to items 43 or 44.

48. A host cell comprising the nucleic acid molecule according to items43 or 44.

49. A method for preparing a homodimeric protein according to any one ofitems 1-42, the method comprising

-   -   a) transfecting the nucleic acid molecule according to items 43        or 44 into a cell population;    -   b) culturing the cell population;    -   c) collecting and purifying the homodimeric protein expressed        from the cell population.

50. A vaccine against a cancer or an infectious disease comprising animmunologically effective amount of a homodimeric protein according toany one of items 1-42 or nucleic acid molecule according to items 43 or44 encoding the monomeric protein which can form the homodimericprotein, wherein said vaccine is able to trigger both a T-cell- andB-cell immune response and wherein said homodimeric protein contain anantigenic unit specific for said cancer or infectious disease.

51. The vaccine according to item 50 further comprising apharmaceutically acceptable carrier and/or adjuvant.

52. The vaccine according to items 50 or 51, wherein said cancer ismultiple myeloma or lymphoma, malignant melanoma, HPV induced cancers,prostate cancer, breast cancer, lung cancer, ovarian cancer, and/orliver cancer.

53. The vaccine according to item 50 or 51, wherein said infectiousdisease is selected from the list consisting of tuberculosis, Influenza,Herpes, CMV, HPV, HBV, HIV, brucellosis, and/or HSV-2.

54. A method of treating a cancer or an infectious disease in a patient,the method comprising administering to the patient in need thereof, ahomodimeric protein according to any one of items 1-42, or the nucleicacid molecule according to items 43 or 44 encoding the monomeric proteinwhich can form the homodimeric protein, wherein said homodimeric proteincontain an antigen unit specific for said cancer or infectious disease.

1. A vector comprising a nucleic acid molecule encoding a monomericprotein, wherein the monomeric protein forms a homodimeric protein oftwo identical amino acid chains, wherein each amino acid chaincomprises: a) a targeting unit comprising an amino acid sequence havingat least 98% sequence identity to the amino acid sequence set forth inpositions 5-70 of SEQ ID NO: 1; b) and an antigenic unit, wherein thetargeting unit and the antigenic unit are connected through adimerization motif and wherein said homodimeric protein provides MHCclass I cross-presentation to CD8+ T cells, and is capable of elicitingan immune response against said antigenic unit.
 2. The vector of claim1, wherein said vector is an expression vector
 3. The vector of claim 1,wherein said nucleic acid molecule is a DNA molecule.
 4. The vector ofclaim 1, wherein the antigenic unit is derived from a bacterium, from avirus, from a HIV antigen or from a HPV antigen.
 5. The vector of claim1, wherein the antigenic unit is selected from the group consisting of:a) a bacterial antigen selected from the list consisting of atuberculosis antigen and a brucellosis antigen; b) a viral antigenselected from the list consisting of influenza virus hemagglutinin (HA),nucleoprotein, M2 antigen and Herpes simplex 2 antigen glycoprotein D;c) a HIV antigen selected from the list consisting of gp120 and Gag; andd) a human papilloma virus antigen selected from the list consisting ofE1, E2, E6, E7, L1 and L2.
 6. The vector of claim 1, wherein theantigenic unit comprises a cancer associated antigen or a cancerspecific antigen.
 7. The vector of claim 6, wherein said cancerassociated antigen is overexpressed on the surface of a cancer cell. 8.The vector of claim 6, wherein said cancer associated or cancer specificantigen is selected from the group consisting of melanoma antigen,prostate cancer antigen, HPV antigen, cervix cancer antigen andidiotypic antigen.
 9. The vector of claim 6, wherein said cancerassociated or cancer specific antigen is a cancer associated or cancerspecific antigen selected from: a) telomerase hTERT; b) a melanomaantigen selected from the list consisting of tyrosinase, TRP-1, andTRP-2; c) prostate cancer antigen PSA; d) a cervix cancer antigenselected from the list consisting of human papilloma virus E1, E2, E4,E6, and E7; and e) fragments of antibodies, wherein the fragments arethe C-terminal scFv derived from the monoclonal Ig produced by myelomaor lymphoma cells, also called the myeloma/lymphoma M component inpatients with B cell lymphoma or multiple myeloma.
 10. The vector ofclaim 3, wherein the dimerization motif comprises a hinge region and animmunoglobulin domain, which are connected through a linker.
 11. Thevector of claim 1, wherein the targeting unit consists of the amino acidsequence of SEQ ID NO:
 1. 12. The vector of claim 11, wherein saidvector is an expression vector
 13. The vector of claim 11, wherein saidnucleic acid molecule is a DNA molecule.
 14. The vector of claim 11,wherein the antigenic unit is derived from a bacterium, from a virus,from a HIV antigen or from a HPV antigen.
 15. The vector of claim 11,wherein the antigenic unit comprises a cancer associated antigen or acancer specific antigen.
 16. The vector of claim 13, wherein thedimerization motif consists of hinge exons h1 and h4 connected through aliker to a CH3 domain of human IgG3.
 14. A pharmaceutical compositioncomprising the vector of claim 1 and a pharmaceutically compatiblecarrier.
 15. A pharmaceutical composition comprising the vector of claim11 and a pharmaceutically compatible carrier.